We hypothesize that in vitro assembly of antigens (Ags) on bacteriophage T4 can be developed as a common platform for construction of next generation multivalent biodefense vaccines. The major goal of this proposal is to test this hypothesis and develop trivalent plague and multivalent plague-anthrax vaccines that can confer protection against Bacillus anthracis and Yersinia pestis, two Category A biodefense pathogens. We have developed a defined in vitro system to array large foreign Ags at high density on phage T4 nanoparticle (120 x 86 nm). Full-length anthrax toxins, as large as 90kDa, are fused to the two non-essential phage T4 outer capsid proteins, Soc (small outer capsid protein; 870 copies) and Hoc (highly antigenic outer capsid protein; 155 copies), over-expressed in E. coli, purified to homogeneity, and displayed on hoc_soc_ capsid through in vitro binding. Multiple Ags and large hetero-oligomeric complexes can be displayed to saturation and the copy number can be controlled. The spatially exposed and symmetrically arrayed T4-rPA particles are highly immunogenic without any adjuvant, eliciting strong PA-specific and lethal toxin neutralizing Antibody (Ab) titers, conferring complete protection to rabbits against 100 LD50 B. anthracis Ames spore challenge. The best vaccine formulations are being tested in a preclinical trial using rhesus macaques. Combining our biochemical, molecular genetic, structural, and aerosol challenge expertise, strategies are designed to develop a novel trivalent T4-plague vaccine comprised of three Yersinia Ags, the capsular Ag, Caf1, the low calcium response V Ag, LcrV, and the Yersinia secretory complex factor, YscF. Inclusion of YscF, a highly conserved needle-forming subunit of type III secretion system that is essential to all virulent Yersinia species, is expected to generate a superior and broadly effective trivalent plague vaccine. The structural disposition and copy number of the capsid-bound plague Ags will be optimized. Combinations of T4-plague Ags, adjuvants, route of delivery (including skin patch), will be tested for Ab and cellular immune responses and protection against challenge with the capsular strain Y. pestis CO92, one of the most lethal plague strains. Efficacy of protection will be assessed in a new Brown Norway rat aerosol challenge model. The best T4-anthrax and T4-plague vaccines will be combined to create a novel multivalent anthrax-plague vaccine. The efficacy of this vaccine for protection against both inhalation anthrax and pneumonic plague will be tested in rhesus macaque aerosol challenge in model.